US008859556B2

(12) United States Patent (10) Patent No.: US 8,859,556 B2 Svensson et al. (45) Date of Patent: Oct. 14, 2014

(54) TRPA1 RECEPTOR ANTAGONIST OTHER PUBLICATIONS (75) Inventors: Mats Svensson, Sodertalje (SE); Dirk Bandell et al., “Noxious Cold Ion Channel TRPAl Is Activated by Weigelt, Ronninge (SE) Pungent Compounds and Bradykinin’. Neuron 2004, 41(6), 849 857. (73) Assignee: Acturum Life Science AB, Solna (SE) Bautista et al. “TRPAl Mediates the Inflammatory Actions of Envi ronmental Irritants and Proalgesic Agents”. Cell 2006, 124(6), 1269 (*) Notice: Subject to any disclaimer, the term of this 1282. patent is extended or adjusted under 35 Jaquemar et al., “An Ankyrin-like Protein with Transmembrane U.S.C. 154(b) by 0 days. Domains Is Specifically Lost after Oncogenic Transformation of Human Fibroblasts”, J Biol Chem 1999, 274(11), 7325-7333. (21) Appl. No.: 13/878,862 Kremeyer et al. "A Gain-of-Function Mutation in TRPAl Causes Familial Episodic Pain Syndrome'. Neuron 2010, 66(5), 671-680. (22) PCT Filed: Oct. 11, 2011 Kwan et al., “TRPA1 Contributes to Cold, Mechanical, and Chemical Nociception but Is Not Essential for Hair-Cell Transduction”, Neu (86). PCT No.: PCT/SE2O11AOS1213 ron 2006, 50(2), 277-289. McGaraughty et al. “TRPA1 modulation of spontaneous and S371 (c)(1), mechanically evoked firing of spinal neurons in uninjured, (2), (4) Date: Jun. 19, 2013 osteoarthritic, and inflamed rats'. Molecular Pain 2010, 6:14. McNamara et al. “TRPA1 mediates formalin-induced pain'. Proc (87) PCT Pub. No.: WO2012/050512 Natl AcadSci USA 2007, 104(33), 13525-13530. PCT Pub. Date: Apr. 19, 2012 Primary Examiner — Kahsay Habte (65) Prior Publication Data (74) Attorney, Agent, or Firm — Baker & Hostetler LLP US 2014/0148466A1 May 29, 2014 (57) ABSTRACT Related U.S. Application Data A compound of Formula (I), pharmaceutically acceptable salts thereof, enantiomers, or mixtures thereof: pharmaceuti (60) Provisional application No. 61/392,130, filed on Oct. cal compositions containing said compounds, enantiomers or 12, 2010. mixtures; processes for making said compounds, enanti (51) Int. Cl. omers or mixtures; the use of said compounds, enantiomers or CO7D 40/4 (2006.01) mixtures; and medicaments containing the same for treatment C07D 213/74 (2006.01) of pain and other conditions; and methods of treating pain and A6 IK3I/44 (2006.01) other conditions with the same. (52) U.S. Cl. USPC ...... 514/253.01:544/360 (58) Field of Classification Search (I) USPC ...... 544/360; 514/253.01 See application file for complete search history. (56) References Cited U.S. PATENT DOCUMENTS

2009,0176883 A1 7/2009 Perner et al. 2009/0325987 A1 12/2009 Muthuppalniappan et al. FOREIGN PATENT DOCUMENTS

WO WO2005/O12287 2, 2005 WO WO2OO7 O73505 6, 2007 WO WO2008/007103 1, 2008 WO WO2010/075353 T 2010 7 Claims, 2 Drawing Sheets U.S. Patent Oct. 14, 2014 Sheet 1 of 2 US 8,859,556 B2

Figure 1.

Counts

1 OOO

500

10 20 30 Position (2Theta) U.S. Patent Oct. 14, 2014 Sheet 2 of 2 US 8,859,556 B2

Figure 2. AbS. 1) Experimental VCD spectrum of example 6 | -- /N ^\ r -\'v. \ ^- A ------/A \- y V

;—-y 2) Inversion - Of calculated VCD?'--/--/ spectrum (4)N/--- ...... A.\J n \W...... /N------Y...... --...... /. . . . :

3) Experimental VCD spectrum of example 7 i

--- rt-:''------,------...-- i ''.

4) Calculated VCD Spe trum for fragment (II) y------W ---, r - \/\----, | Ay A- \- ill- v/-- --- 1700 1600 1500 1400 1300 1200 1100 1000 900 Wavenumbers (cm)

VCD Experimental and Calculated Spectra. US 8,859,556 B2 1. 2 TRPA1 RECEPTOR ANTAGONST pensity to work as an analgesic in vivo. An object of the present invention is therefore to provide new, improved and FIELD OF THE INVENTION useful analgesics. The present invention relates to antagonists of the transient SUMMARY OF THE INVENTION receptor potential (TRP) family of ion channels. The present Non-selective cation channel TRPA1 antagonists are invention also provides compositions comprising Such described. The described antagonists can be used for treating antagonists and methods therewith for treating diseases medi pain and other conditions. Medical conditions which are ated by the transient receptor potential (TRP) family of ion mediated by TRPA1 antagonists include asthma, pertussis channels. Particularly, the present invention is related to non 10 and nicotine addiction. selective cation channel TRPA1 antagonist compounds use ful for treating pain and other specific disorders. BRIEF DESCRIPTION OF THE DRAWINGS BACKGROUND FIG. 1 shows an X-Ray Powder Diffraction (XRPD) pat 15 tern for the compound of Example 6. The transient receptor potential (TRP) family of ion chan FIG. 2 shows VCD Spectra for compounds. nels are often found to be sensors of multiple chemical and physical stimuli (temperature, Smell, taste and noxious DETAILED DESCRIPTION OF THE INVENTION chemicals). Particularly, the non-selective cation channel TRPA1 (transient receptor potential ankyrin-repeat 1), origi The present invention thus provides new antagonists of the nally cloned as an ankyrin-like protein (Jaquemar et al 1999, non-selective cation channel TRPA1. The new antagonists J Biol Chem 274:7325-7333), is activated by multiple pun can be used for, amongst other things, treating pain. Further gent and pro-algesic compounds (Bandell et al 2004, Neuron medical conditions which are mediated by TRPA1 antago 41: 849-857). Recent reviews on TRPA1 as pharmaceutical nists include asthma, pertussis and nicotine addiction. target have been published by Rechet al. (Future Med. Chem. 25 In a first aspect, the present invention provides a compound 2010: 843-838) and by Baraldi et al (J. Med. Chem. 2010, of Formula (I), a pharmaceutically acceptable salt thereof, 5085-2107). TRPA1 is highly expressed in a subset of C-fiber enantiomer, or mixture thereof: nociceptors in the peripheral nerve system (Kobayashi et al (I) 2005, J. Comp Neurol 493(4):596-606). When activated TRPA1 permits the conduction ofcations (primarily Ca" and 30 2 Na") from the extra cellular environment into the cell, thereby depolarizing the membrane potential and affecting calcium homeostasis in the primary afferents. Depolarization primary nerve terminals lead to action potential firing and conse quently increased pain sensation and hyperalgesia in man and 35 rodent experimental models (Jiang and Gebhart 1998, Pain 77(3):305-13; Cervero and Laird 1996, Pain 68(1):13–23). The pungent ingredient in mustard oil: allyl isothiocyanate (AITC), concentration dependently activates TRPA1 in vitro measured both on sodium current and calcium influx. Further, 40 AITC also excites small diameter afferent fibers (Reeh 1986, It will be understood that as a compound of the present Brain Res 384:42-50) and indeed topical application of AITC invention contains one chiral centers, the compound may induces pain and hyperalgesia in man (Namer et al 2005, exist in, and be isolated as, enantiomeric forms, or as a race Neuroreport 16(9):955-959). Recently TRPA1 knock out mic mixture. The present invention includes any possible (KO) mice were described to have lost AITC sensitivity and 45 enantiomers, racemates or mixtures thereof, of a compound display severely impaired bradykinin pain response signal of Formula (I). The optically active forms of the compound of ling (Kwan 2006, Neuron 50(2):277-289; Bautista 2006, Cell the invention may be prepared, for example, by chiral chro 124(6):1269-1282). Formalin, which is a mixture of metha matographic separation of a racemate, by synthesis from opti nol, water and formaldehyde is a widely used rodent model cally active starting materials or by asymmetric synthesis for evaluating analgesic compounds in vivo. TRPA1 is acti 50 based on the procedures described thereafter. vated by formaldehyde in vitro and recently it was shown that One embodiment of the present invention is the (R) enan TRPA1 KO mice almost have their response to formalin/ tiomer of a compound of Formula (I) having the following formaldehyde abolished (McNamara et al 2007, Proc Natl Structure: AcadSci USA 104(33):13525-13530). A Hydra Biosciences proprietary compound (HCO30031) is a TRPA1 antagonist in 55 vitro and has been shown to alleviate the formalin induced 2 pain behaviour in a dose dependant way (McNamara 2007, Proc Natl Acad Sci USA 104(33):13525-13530 and WO2007/073505A2). An Abbott proprietary compound (US l 2009/0176883 and McGaraughty et al., Molecular Pain 2010 r^- 6:14) with in vitro effect on TRPA1 showed to have in vivo 60 N Nu O effect in a rat osteoarthritis pain model as well as a number of Glenmark proprietary compounds (US 2009/0325987) showed in several in vivo pain models. It has also been shown l that a mutation in TRPA1 causes FEPS (“Familial Episodic Pain Syndrome', Kremeyeretal, Neuron 2010, 66:671-680). 65 It is therefore suggested that in vitro inhibition of the TRPA1 It will also be understood that a compound of the present calcium and Sodium influx will determine a compound's pro invention may exist in Solvated, for example hydrated, as well US 8,859,556 B2 3 4 as unsolvated forms. It will further be understood that the tive pain, inflammatory pain, central pain, central and periph present invention encompasses all such solvated forms of the eral neuropathic pain, central and peripheral neurogenic pain, compound of the Formula (I). central and peripheral neuralgia, chronic tendinitis, low back Within the scope of the invention are also salts of the pain, postoperative pain, peripheral neuropathy, visceral compound of the Formula (I). Generally, pharmaceutically pain, pelvic pain, allodynia, anesthesia dolorosa, causalgia, acceptable salts of compound of the present invention may be dysesthesia, fibromyalgia, hyperalgesia, hyperesthesia, obtained using standard procedures well known in the art, for hyperpathia, ischemic pain, Sciatic pain, pain associated with example by reacting a sufficiently basic compound, for cystitis, including but not limited to interstitial cystitis, pain example an alkylamine with a suitable acid, for example, HCl associated with multiple Sclerosis, pain associated with or acetic acid, to afford a physiologically acceptable anion. It 10 may also be possible to make a corresponding alkali metal arthritis, pain associated with osteoarthritis, pain associated (such as sodium, potassium, or lithium) or an alkaline earth with rheumatoid arthritis, and pain associated with cancer. metal (Such as a calcium) salt by treating a compound of the The present invention further provides a compound of For present invention having a suitably acidic proton, such as a mula (I) for the treatment of asthma, pertussis (persistent carboxylic acid or a with one equivalent of an alkali 15 cough), nicotine addiction and of various pain conditions metal or alkaline earth metal hydroxide or alkoxide (such as Such as acute and chronic pain disorders including but not the ethoxide or methoxide), or a Suitably basic organic amine limited to widespread pain, localized pain, nociceptive pain, (such as choline or meglumine) in anaq medium, followed by inflammatory pain, central pain, central and peripheral neu conventional purification techniques. ropathic pain, central and peripheral neurogenic pain, central In one embodiment, a compound of Formula (I) above may and peripheral neuralgia, chronic tendinitis, low back pain, be converted to a pharmaceutically acceptable salt or Solvate postoperative pain, peripheral neuropathy, visceral pain, pel thereof, particularly, an acid addition salt such as a hydro vic pain, allodynia, anesthesia dolorosa, causalgia, dysesthe chloride, hydrobromide, phosphate, acetate, fumarate, male sia, fibromyalgia, hyperalgesia, hyperesthesia, hyperpathia, ate, tartrate, citrate, methanesulphonate or p-toluenesulpho ischemic pain, Sciatic pain, pain associated with cystitis, nate. 25 including but not limited to interstitial cystitis, pain associ A compound of the present invention is contemplated to be ated with multiple Sclerosis, pain associated with arthritis, useful in therapy, especially for the therapy asthma, pertussis pain associated with osteoarthritis, pain associated with rheu (persistent cough), nicotine addiction and of various pain matoid arthritis, and pain associated with cancer. conditions including, but not limited to, acute and chronic A further aspect of the invention is a method for the treat pain disorders including but not limited to widespread pain, 30 ment of a Subject Suffering from any of the conditions dis localized pain, nociceptive pain, inflammatory pain, central cussed above, whereby an effective amount of a compound pain, central and peripheral neuropathic pain, central and according to the Formula (I) above, or pharmaceutically peripheral neurogenic pain, central and peripheral neuralgia, acceptable salt or Solvate thereof, is administered to a patient chronic tendinitis, low back pain, postoperative pain, periph in need of Such treatment. eral neuropathy, visceral pain, pelvic pain, allodynia, anes 35 Thus, the invention provides a method for treating the thesia dolorosa, causalgia, dysesthesia, fibromyalgia, hyper specific medical indications set out above, wherein an effec algesia, hyperesthesia, hyperpathia, ischemic pain, Sciatic tive amount of a compound of Formula (I) or pharmaceuti pain, pain associated with cystitis, including but not limited to cally acceptable salt or solvate thereof, is administered to a interstitial cystitis, pain associated with multiple Sclerosis, patient in need of Such treatment. pain associated with arthritis, pain associated with osteoar 40 The present invention also provides a compound of For thritis, pain associated with rheumatoid arthritis, and pain mula (I) for use as a medicament in therapy. associated with cancer. In the context of the present specification, the term In use for therapy in a warm-blooded animal such as a “therapy' also includes “prophylaxis' unless there are spe human, a compound of the invention may be administered in cific indications to the contrary. The term “therapeutic' and the form of a conventional pharmaceutical composition by 45 “therapeutically should be construed accordingly. The term any route including orally, intramuscularly, Subcutaneously, “therapy” within the context of the present invention further topically, intranasally, intraperitoneally, intrathoracically, encompasses to administer an effective amount of a com intravenously, epidurally, intrathecally, transdermally, intrac pound of the present invention, to mitigate either a pre-exist erebroventricularly and by injection into the joints. ing disease state, acute or chronic, or a recurring condition. In one embodiment of the invention, the route of adminis 50 This definition also encompasses prophylactic therapies for tration may be intravenous, topical, intradermal or intramus prevention of recurring conditions and continued therapy for cular. chronic disorders. The dosage will depend on the route of administration, the The compounds of Formula (I) have activity as pharma severity of the disease, age and weight of the patient and other ceuticals, in particular as inhibitors (antagonists) of TRPA1. factors normally considered by the attending physician, when 55 More particularly, the TRPA1 inhibitors of the present inven determining the individual regimen and dosage level at the tion are useful in therapy, especially for relief of various pain most appropriate for a particular patient. conditions such as acute and chronic pain disorders including The present invention provides the use of a compound of but not limited to widespread pain, localized pain, nocicep Formula (I) or a pharmaceutically acceptable salt or Solvate tive pain, inflammatory pain, central pain, central and periph thereof, as hereinbefore defined in the manufacture of a medi 60 eral neuropathic pain, central and peripheral neurogenic pain, cament for use in therapy. central and peripheral neuralgia, chronic tendinitis, low back In particular medical indications, the present invention pain, postoperative pain, peripheral neuropathy, visceral provides the use of a compound of Formula (I) for the manu pain, pelvic pain, allodynia, anesthesia dolorosa, causalgia, facture of a medicament for the treatment of asthma, pertussis dysesthesia, fibromyalgia, hyperalgesia, hyperesthesia, (persistent cough), nicotine addiction and of various pain 65 hyperpathia, ischemic pain, Sciatic pain, pain associated with conditions such as acute and chronic pain disorders including cystitis, including but not limited to interstitial cystitis, pain but not limited to widespread pain, localized pain, nocicep associated with multiple Sclerosis, pain associated with US 8,859,556 B2 5 6 arthritis, pain associated with osteoarthritis, pain associated etable oil or polyethylene glycol. Hard gelatine capsules may with rheumatoid arthritis, and pain associated with cancer. contain granules of the compound using either the above A compound according to Formula (I) may also be used in mentioned excipients for tablets. Also liquid or semisolid treatment of ophthalmological disorders such as retinopa formulations of the compound of the invention may be filled thies, diabetic retinopathies and glaucoma and treatment of 5 into hard gelatine capsules. pain associated with Such disorders. Liquid preparations for oral application may be in the form The compounds according to Formula (I) may also be used of syrups or Suspensions, for example, Solutions containing for ophthalmic treatment or for use as counter-agent for "riot the compound of the invention, the balance being Sugar and a control agents' such as CS or CR and for treating subjects mixture of ethanol, water, glycerol and propylene glycol. Suffering from the effects of Such agents by administering a 10 Optionally such liquid preparations may contain colouring therapeutically effective amount of a compound according to agents, flavouring agents, saccharine and/or carboxymethyl Formula (I). For the above-mentioned therapeutic uses the dosage cellulose as a thickening agent or other excipients known to administered will, of course, vary with the compound those skilled in art. employed, the mode of administration, the treatment desired 15 The compound of the invention may also be administered and the disorder indicated. The daily dosage of the compound in conjunction with other compounds used for the treatment of the invention may be in the range from 0.05 mg/kg to 100 of the above conditions. mg/kg. In a further embodiment, a compound of the present inven The compound of formula (I) and pharmaceutically tion, or a pharmaceutical composition or formulation com acceptable salts thereof may be used on their own but will prising a compound of Formula (I) is administered concur generally be administered in the form of a pharmaceutical rently, simultaneously, sequentially or separately with composition in which the formula (I) compound/salt (active another pharmaceutically active compound or compounds ingredient) is in association with a pharmaceutically accept selected from the following: able adjuvant, diluent or carrier. Conventional procedures for (i) such as , , amox the selection and preparation of Suitable pharmaceutical for 25 apine, bupropion, citalopram, clomipramine, desipramine, mulations are described in, for example, "Pharmaceuticals— dulloxetine, elZasonan, escitalopram, fluvoxamine, The Science of Dosage Form Designs, M. E. Aulton, fluoxetine, gepirone, imipramine, ipsapirone, maprotiline, Churchill Livingstone, 1988. nortriptyline, , paroxetine, phenelzine, protrip Depending on the mode of administration, the pharmaceu tyline, , reboxetine, robalZotan, Sertraline, Sibutra tical composition will preferably comprise from 0.05 to 99% 30 mine, thionisoxetine, tranylcypromaine, , trimi w (percent by weight), more preferably from 0.05 to 80% w, pramine, Venlafaxine and equivalents and pharmaceutically still more preferably from 0.10 to 70% w, and even more active isomer(s) and metabolite(s) thereof. preferably from 0.10 to 50% w, of active ingredient, all per (ii) atypical including for example centages by weight being based on total composition. The and pharmaceutically active isomer(s) and metabolite(s) present invention also provides a pharmaceutical composi 35 thereof. tion comprising a compound of formula (I) or a pharmaceu (iii) antipsychotics including for example amisulpride, arip tically acceptable salt thereof as hereinbefore defined, in iprazole, asenapine, benzisoxidil, bifeprunoX, carbam association with a pharmaceutically acceptable adjuvant, azepine, clozapine, , debenzapine, dival diluent or carrier. proex, dulloxetine, , haloperidol, illoperidone, The invention further provides a process for the prepara 40 lamotrigine, loxapine, mesoridazine, , paliperi tion of a pharmaceutical composition of the invention which done, , perphenazine, phenothiazine, phenylbu comprises mixing a compound of formula (I) or a pharma tylpiperidine, pimozide, prochlorperazine, , ceutically acceptable salt thereofas hereinbefore defined with sertindole, Sulpiride, , , , tri a pharmaceutically acceptable adjuvant, diluent or carrier. fluoperazine, , Valproate, Valproic acid, , The pharmaceutical compositions may be administered 45 Zotepine, Ziprasidone and equivalents and pharmaceutically topically (e.g. to the skin) in the form, e.g., of creams, solu active isomer(s) and metabolite(s) thereof. tions or Suspensions; or systemically, e.g. by oral administra (iv) anxiolytics including for example alnespirone, aza tion in the form of tablets, capsules, syrups, powders or gran pirones, , such as adinazolam, ules; or by parenteral administration in the form of solutions alprazolam, balezepam, bentazepam, bromazepam, broti or Suspensions; or by Subcutaneous administration; or by 50 Zolam, buspirone, , cloraZepate, chlordiazep rectal administration in the form of Suppositories; or trans oxide, cyprazepam, diazepam, , , dermally. For oral administration the compound of the inven fenobam, , , fosazepam, , tion may be admixed with an adjuvant or a carrier, for , , , , example, lactose, Saccharose, Sorbitol, mannitol; a starch, for oxazepam, prazepam, , reclazepam, tracaZolate, example, potato starch, corn starch or amylopectin; a cellu 55 trepipam, , , uldazepam, Zolazepam and lose derivative; a binder, for example, gelatine or polyvi equivalents and pharmaceutically active isomer(s) and nylpyrrolidone; and/or a lubricant, for example, magnesium metabolite(s) thereof. Stearate, calcium Stearate, polyethylene glycol, a wax, paraf (v) anticonvulsants including for example carbamazepine, fin, and the like, and then compressed into tablets. If coated clonazepam, ethoSuximide, felbamate, fosphenyloin, gabap tablets are required, the cores, prepared as described above, 60 entin, lacosamide, lamotrogine, levetiracetam, oXcarba may be coated with a concentrated Sugar Solution which may Zepine, , phenyloin, pregabaline, rufinamide, contain, for example, gum arabic, gelatine, talcum and tita topiramate, valproate, vigabatrine, Zonisamide and equiva nium dioxide. Alternatively, the tablet may be coated with a lents and pharmaceutically active isomer(s) and metabolite(s) Suitable polymer dissolved in a readily Volatile organic Sol thereof. Vent. 65 (vi) Alzheimer's therapies including for example donepezil, For the preparation of softgelatine capsules, the compound rivastigmine, galantamine, memantine, and equivalents and of the invention may be admixed with, for example, a Veg pharmaceutically active isomer(s) and metabolite(s) thereof. US 8,859,556 B2 7 8 (vii) Parkinson's therapies including for example deprenyl, Z-gradients, or a Bruker Avance 600 NMR spectrometer L-dopa, Requip, Mirapex. MAOB inhibitors such as selegine equipped with a 5 mm BBI probe head with Z-gradients. and rasagiline, comP inhibitors such as Tasmar, A-2 inhibi Unless specifically noted in the examples, spectra were tors, dopamine reuptake inhibitors, NMDA antagonists, recorded at 400 MHz for proton, 376 MHz for F and 100 Nicotine agonists, Dopamine agonists and inhibitors of neu MHz for C. ronal nitric oxide synthase and equivalents and pharmaceuti Alternatively, H and 'C NMR spectra were recorded at cally active isomer(s) and metabolite(s) thereof. 400 MHz for proton and 100 MHz for 'C on a Varian Mer (viii) migraine therapies including for example almotriptan, cury Plus 400 NMR Spectrometer equipped with a Varian 400 amantadine, bromocriptine, butalbital, cabergoline, dichlo ATB PFG probe. ralphenaZone, dihydroergotamine, eletriptan, froVatriptan, 10 The following reference signals were used: the middle line lisuride, naratriptan, pergolide, pizotiphen, pramipexole, of DMSO-d 8 2.50 (1H), & 39.51 (13C); the middle line of rizatriptan, ropinirole, Sumatriptan, Zolmitriptan, Zomitrip CDOD 83.31 (1H) or 849.15 (13C). CDC1 & 7.26 (1H) and tan, and equivalents and pharmaceutically active isomer(s) the middle line of CDC1 & 77.16 (13C) (unless otherwise and metabolite(s) thereof. indicated). NMR spectra are reported from low to high field. (ix) stroke therapies including for thrombolytic therapy with 15 Alternatively, all deuterated solvents contained typically eg activase and desmoteplase, abciximab, citicoline, clopi 0.03% to 0.05% v/v tetramethylsilane, which was used as the dogrel, eptifibatide, minocycline, and equivalents and phar reference signal (set at 8 0.00 for both Hand 'C). maceutically active isomer(s) and metabolite(s) thereof. Mass spectra were recorded on a Waters LCMS consisting (X) urinary incontinence therapies including for example of an Alliance 2795 (LC), Waters PDA 2996 and a ZQ single darafenacin, falvoxate, Oxybutynin, propiverine, robalZotan, quadrupole mass spectrometer or Waters Micromass ZQ Solifenacin, tolterodine and equivalents and pharmaceutically detector at 120°C. The mass spectrometer was equipped with active isomer(s) and metabolite(s) thereof. an electrospray ion source (ESI) operated in a positive or (xi) neuropathic pain therapies including for example negative ion mode. The capillary Voltage was 3 kV and cone lidocain, capsaicin, and anticonvulsants such as , Voltage was 30 V. The mass spectrometer was scanned , and antidepressants such as dulloxetine, Venlafax 25 between m/Z 100-700 or m/z. 100-1000 with a scan time of 0.3 ine, amitriptyline, kilomipramine, and equivalents and phar S. Separations were performed on either Waters X-Terra MS maceutically active isomer(s) and metabolite(s) thereof. C8 (3.5um, 50 or 100mmx2.1 mm i.d.) or an ACE3AQ (100 (xii) nociceptive pain therapies such as paracetamol, NSAIDs mmx2.1 mm i.d.) obtained from ScantecLab. Flow rates were and coxibs, such as celecoxib, etoricoxib, lumiracoxib, Val regulated to 1.0 or 0.3 mL/min, respectively. The column decoxib, parecoxib, diclofenac, loxoprofen, naproxen, keto 30 temperature was set to 40°C. A linear gradient was applied profen, ibuprofen, nabumeton, meloxicam, piroxicam and using a neutral or acidic mobile phase system, starting at such as , , buprenorfin, tramadol, 100% A (A: 95:5 10 mM NHOAc:MeCN, or 95:58 mM and equivalents and pharmaceutically active isomer(s) and HCOOH:MeCN) ending at 100% B (MeCN). metabolite(s) thereof. Mass spectra also were recorded on a Waters LCMS con (xiii) therapies including for example agomelatine, 35 sisting of an Alliance 2690 Separations Module, Waters 2487 , alonimid, , benzoctamine, butabar Dual 1 Absorbance Detector (220 and 254 nm) and a Waters bital, capuride, chloral, cloperidone, clorethate, dexclamol. ZQ single quadrupole mass spectrometer. The mass spec , , , halazepam, hydrox trometer was equipped with an electrospray ion source (ESI) yZine, , , mephobarbital, methaqua operated in a positive or negative ion mode. The capillary lone, midaflur, , , phenobarbital, 40 voltage was 3 kV and cone voltage was 30 V. The mass , ramelteon, roletamide, , , Zale spectrometer was scanned between m/z, 97-800 with a scan plon, and equivalents and pharmaceutically active time of 0.3 or 0.8 s. Separations were performed on a Chro isomer(s) and metabolite(s) thereof. molith Performance RP-18e (100x4.6 mm). A linear gradient (xiv) mood stabilizers including for example carbamazepine, was applied starting at 95% A (A: 0.1% HCOOH (aq)) end divalproex, gabapentin, lamotrigine, lithium, olanzapine, 45 ing at 100% B (MeCN) in 5 min. Flow rate: 2.0 mL/min. quetiapine, Valproate, valproic acid, Verapamil, and equiva Alternatively, Ultra Pressure (UP) LCMS analyses were lents and pharmaceutically active isomer(s) and metabolite(s) performed on an Waters Acquity UPLC system consisting of thereof. Such combination products employ the compound of a Acquity Autosampler, Acquity Sample Organizer, Acquity this invention within the dosage range described herein and Column Manager, Acquity Binary Solvent Manager, Acquity the other pharmaceutically active compound or compounds 50 UPLC PDA detector and a Waters SQ Detector. The mass within approved dosage ranges and/or the dosage described in spectrometer was equipped with an electrospray ion Source the publication reference. (ES) operated in positive and negative ion mode. The capil Method of Preparation, Experimental Work lary voltage was set to 3.0 kV and the cone voltage to 30 V. General Methods respectively. The mass spectrometer was scanned between All solvents used were analytical grade and commercially 55 m/Z 100-600 with a scan time of 0.105 s. The diode array available anhydrous solvents were routinely used for reac detector scanned from 200-400 nm. The temperature of the tions. Reactions were typically run under an inert atmosphere Column Manager was set to 60° C. Separation was performed of nitrogen or argon. ona Acquity column, UPLC BEH, C18 1.7 LM run at a flow 'H, °F and 'C NMR spectra were recorded on a Varian rate of 0.5 mL/min. A linear gradient was applied Starting at Unity+400 NMR Spectrometer equipped with a 5 mm BBO 60 100% A(A: 10 mMNHOAc in 5% MeCN) ending at 100% probe-head with Z-gradients, or a Varian Gemini 300 NMR B (B: MeCN) after 1.3 min then 100% B for 0.6 min. ESpos/ spectrometer equipped with a 5 mm BBI probe head, or a ESneg, m/z. 100-600. Bruker Avance 400 NMR spectrometer equipped with a 60 ul Compound identification was also performed on a GC-MS dual inverse flow probe head with Z-gradients, or a Varian system Supplied by Agilent Technologies, consisting of a Mercury Plus 400NMR Spectrometer equipped with a Varian 65 6890NG1530NGC, a G2614AAutosampler, G2613A injec 400 ATB PFG probe, or a Bruker DPX400 NMR spectrom tor and a G2589N mass spectrometer. The columnused was a eter equipped with a 4-nucleus probe head equipped with VF-5 MS, ID 0.25mmx30 m, 0.25um (Varian Inc.). A linear US 8,859,556 B2 10 temperature gradient was applied starting at 70° C. (hold 1 condition of a mixture of EtOH and CO was applied at flow min) and ending at 300° C. (hold 1 min), 25°C/min. The rate 50.0 mL/min. The UV detector scanned at 220 nm. The mass spectrometer was equipped with a chemical ionisation UV signal determined the fraction collection. (CI) ion source and the reactant gas was methane. The mass Compounds have been named using either ACD/Name, spectrometer scanned between m/z 50-500 and the scan speed version 10.06, software from Advanced Chemistry Develop was set to 3.21 scan/s. Solvent delay was set from 0 minto 2.0 ment, Inc. (ACD/Labs), Toronto ON. Canada, www.acdlabs. 1. com, or Lexichem, version 1.4. Software from OpenEye. HPLC analyses were performed on an Agilent HP1000 system consisting of G1379A Micro Vacuum Degasser, ABBREVIATIONS G1312A Binary Pump, G1367A Well plate autosampler, 10 G 1316A Thermostatted Column Compartment and G 1315B Abs Absorption Diode Array Detector. Column: X-Terra MS, Waters, 3.0x aq aqueous 100 mm, 3.5 Lum. The column temperature was set to 40°C. CAS Chemical Abstracts Service and the flow rate to 1.0 mL/min. The Diode Array Detector CDI 1.1'-carbonyldiimidazole was scanned from 210-300 nm, step and peak width were set 15 DCM dichloromethane to 2 nm and 0.05 min, respectively. A linear gradient was DIPEAN,N-diisopropylethylamine applied, starting at 100% A (A: 95:5 10 mM NHOAc: DMF dimethylformamide MeCN) and ending at 100% B (B: MeCN), in 4 min. DMSO dimethyl sulfoxide HPLC analyses were also performed with a Gynkotek EDCI 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide P580 HPG consisting of gradient pump with a Gynkotek ES electro spray UVD 170S UV-vis.-detector equipped with a Chromolith ESI electro spray ionization Performance RP column (C18, 100 mmx4.6 mm). The col eq equivalent(s) umn temperature was set to 25° C. A linear gradient was GC gas chromatography applied using Mecn/0.1 trifluoroacetic acid in MilliO water, h hour(s) run from 10% to 100% MeCN in 5 min. Flow rate: 3 mL/min. 25 HCl hydrochloric acid, hydrochloride Chiral purity analysis was run on a SFC Berger Analytix HPLC high-performance liquid chromatography system with Agilent 1100 PDA detector. The column tem IPC In-process control perature was set to 50° C. An isocratic condition a mixture of M Molar (moles per liter) EtOH and CO was applied at flow rate 2.0 mL/min. The PDA min minute(s) was scanned from 190-600 nm and 220 nm was extracted for 30 MS mass spectrometry purity determination. NMR nuclear magnetic resonance Chiral HPLC analyses were alternatively performed on a prep. Preparative Gilson chiral system Column: Chiralpak AD-H, 4.6*250 mm: rt room temperature 5 um Mobile phase: 100% EtOH Flow rate: 0.8 mL/min. Rt retention time Optical rotation was determined with a PDR-Chiral laser 35 SFC Supercritical fluid chromatography polarimeter. Microwave heating was performed in a single T, Jacket temperature mode microwave cavity producing continuous irradiation at T, Internal temperature 2450 MHZ. TEA triethylamine Thin layer chromatography (TLC) was performed on TLC thin layer chromatography Merck TLC-plates (Silica gel 60Fs) and UV visualized the 40 THF tetrahydrofuran spots. TLC thin layer chromatography Flash chromatography was performed on a Combi FlashR) UV ultraviolet CompanionTM using RedisepTM normal-phase flash columns VCD Vibrational Circular Dichroism or using Merck Silica gel 60 (0.040-0.063 mm). Typical sol Starting Materials vents used for flash chromatography were mixtures of chlo 45 roform/methanol, dichloromethane/methanol, heptane/ethyl Example 1 acetate, /methanol/ammonia (aq.) and dichlo rormethane/methanol/NH (aq.). tert-Butyl 1-(methoxy(methyl)amino)-3.3-dimethyl Preparative chromatography was run ona Waters auto puri 1-oxobutan-2-ylcarbamate fication HPLC with a diode array detector. Column: XTerra 50 MS C8, 19x300mm, 10um. Narrow gradients with MeCN/ (95:50.1M NHOAc:MeCN) were used at a flow rate of 20 mL/min. O Purification was also achieved on a semi preparative Shi O O madzu LC-8A HPLC with a Shimadzu SPD-10A UV-vis.- 55 detector equipped with a Waters Symmetry(R) column (C18, 5 y r N1 N. um, 100 mmx19 mm). Narrow gradients with Mecn/0.1% O trifluoroacetic acid in MilliO Water were used at a flow rate of 10 mL/min. Alternatively, purification was achieved on a preparative 60 Method 1 Gilson 281 (Gilson Pump 322) HPLC with a Gilson 156 To a solution of 2-(tert-butoxycarbonylamino)-3,3-dim UV-detector equipped with a Waters Sunfire column (150 ethylbutanoic acid (CAS 102185-35-3, 500 mg, 2.16 mmol) mmx21.2 mm). Narrow gradients with Mecn/0.1% formic in DCM (5 mL) was added EDCI (CAS25952-53-8, 497 mg, acid in water were used at a flow rate of 15 mL/min. 2.59 mmol) and stirred for 10 min at rt. A solution of NO Chiral preparative chromatography was run on a SFC 65 dimethylhydroxylaminehydrochloride (CAS.6638-79-5, 253 Berger Multigram system with a Knauer K-2501 UV detec mg, 2.59 mmol) and DIPEA (0.429 mL, 2.59 mmol) in DCM tor. The column temperature was set to 35° C. An isocratic (5 mL) was added and the reaction mixture was left to stir at US 8,859,556 B2 11 12 rt over a weekend. The mixture was diluted with DCM (30 Example 2b mL) and extracted with saturated aq NaHCO, (50 mL). The organic phase was washed with water before being dried over tert-Butyl 1-(4-cyanophenyl)-3,3-dimethyl-1-oxobu MgSO and filtered. The solvent was removed in vacuo to tan-2-ylcarbamate give tert-butyl 1-(methoxy(methyl)amino)-3,3-dimethyl-1- 5 oxobutan-2-ylcarbamate (466 mg, 79%). "H NMR (500 MHz, CDC1) 8 ppm 0.98 (s, 9H) 1.43 (s, 9H)2.19-2.28 (m. 1H)3.21 (s.3H)3.78 (s.3H)4.66 (d. 1H). GC-MS (unprotected amino acid) m/z, 130, Rt. 5.82 min. Method 2 10 To a cleaned and dried 10L reactoratT,25°C. was charged N-boc-tert-butylglycin (N-Boc-tert-Leucine, CAS 102185 rol 35-3) (188 g; 0.77 mol: 1.00 eq) and DCM (6.25 L: 35 vol) giving a fine slurry. 1,1'-Carbonylimidazole (208.7 g; 1.3 eq) To a dried and cleaned 10 L cryo reactor was added 4-bro 15 mobenzonitrile (321 g; 1.74 mol; 2.5 eq) and THF (995 mL.; was added under stirring in one portion affording a clear pale 5.0 vol). The stirred mixture was inerted under a nitrogen green Solution after approx 1 min under gas exhaust. The atmosphere and cooled (T, -20°C.). To the cooled mixture at reaction was monitored by GC and after 1 hrt at T 25° C. a T -13°C. Turbo Grignard reagent (Turbo Grignard Reagent second portion of 1,1'-carbonylimidazole (32.1 g; 0.2 eq) was from Chemetall equal to iPrMgCl/LiCl 14% w/w in THF: added and the reaction mixture was left overnight. To the clear 1.81 L; 2.5 eq) was charged under the nitrogen atmosphere reaction mixture N.O-dimethylhydroxylamine (153.7 g; 2.0 while maintaining the temperature below approx -10° C. eq) was added followed by TEA (215 mL 2.0 eq). T, was set during 1 h 14 min. The reaction intermediate was left at to 50° C. and the reaction conversion was followed by GC approx 0°C. for 3 h giving a conversion of >97% (analytical analyses. A second portion of N.O-dimethylhydroxylamine sample quenched with 15% w/waq NHCl). The mixture was (26.9 g; 0.35 eq) and TEA (37 mL: 0.35 eq) was added to the cooled to T, -20° C. and a solution of tert-butyl 1-(methoxy mixture and the reaction was left for 48 h at T, 50° C. (T, 41° 25 (methyl)amino)-3.3-dimethyl-1-oxobutan-2-ylcarbamate C.) giving a conversion of 98.7%. The reaction mixture (ap (191 g; 0.70 mol; 1.0 eq) in THF 496 mL: 2.6 vol) was prox pH 8) was cooled and at T, 20°C. water (2.82 L; 15 vol) charged to the reactor for 28 min at approx-10°C. to -6° C. was charged to the reactor and the resulting mixture was (exothermal reaction). The vessel was rinsed with THF (221 stirred for 10 min. The organic layer was recovered and mL: 1.3 L), the reaction mixture warmed to 20° C. and left washed twice with water (2x2.82 L; 2x15 vol), NaHCO 30 overnight affording a conversion (HPLC) of approx 90%. The (2x2.3 L; 2x12 Vol) and finally with water (2x1.88 L; 2x10 mixture was cooled to 0°C. (T-10) and a solution of potas Vol). The organic layer was concentrated in vacuo at 40° C. sium sodium tartrate (Rochelle salt) (153 g; 1.05 eq) in water giving a yellow residual oil, which was co-evaporated with (10 Vol) was drop wise added during 30 min keeping the toluene (250 mL) giving 295 goily product and left overnight. temperature at 0 to 10°C. giving an orange coloured slurry. T, A crystalline mixture appeared. The mixture was filtered (P3 35 was set to 40°C. and stirring was stopped at T., 30° C. and the sintered disk; 100 mm diameter), washed with toluene (75 phases allowed to separate. The red coloured organic layer mL) and dried affording 138g of white crystalline material. A was recovered and the yellow slurry-like water phase was second crop (158 g) was obtained, dissolved in hot n-heptane extracted twice at T,30°C. withiPrOAC (2x1.91 L:2x10 vol). (250 mL) cooled with an external ice-water bath giving 56 g The organic layers were combined (7.5 L) and washed at T, of pale colored material. Analyses gave of first crop 96.8% 30° C. three times with a brine-water mixture (3+3 vol; 5+5 w/w and of second crop 72% w/w respectively. Total yield 40 vol: 3+3 vol). The washed organic layer was concentrated in (194 g) based on NMR assays was 82%. vacuo at T, 60° C. to approx 1.5 L volume and diluted (iP Example 2a rOAc) to 2 L. The product solution was taken to the next step tert-Butyl 1-(4-chlorophenyl)-3,3-dimethyl-1-oxobu (example 3, method 2) without further purification. tan-2-ylcarbamate 45 Example 3 4-(2-Amino-3,3-dimethylbutanoyl)benzonitrile hydrochloride

50 Yr Cl NH, HCI tert-Butyl 1-(methoxy(methyl)amino)-3,3-dimethyl-1-ox obutan-2-ylcarbamate (Example 1,466 mg, 1.70 mmol) was dissolved in dry THF (10 mL). (4-Chlorophenyl)magnesium 55 (CAS873-77-8, 1.0 M in THF, 6.79 mL, 6.79 mmol) 2 was then added dropwise at rt. After stirring at rt overnight the reaction was quenched with NHCl and extracted with EtOAc Method 1 (2x50 mL). The combined organic phases were washed once tert-Butyl 1-(4-chlorophenyl)-3,3-dimethyl-1-oxobutan with water, dried over MgSO and filtered. After removal of 60 2-ylcarbamate (Example 2a, 2.30g, 7.06 mmol) and Zn(CN), the solvent the product was purified on a silica gel column (0.87g, 7.4 mmol) were dissolved in DMF (20 mL) under N2 eluted with heptane:EtOAc 10-20% to give tert-butyl 1-(4- (g). Pd(PPh) (0.86g, 0.74 mmol) was added and the mixture chlorophenyl)-3,3-dimethyl-1-oxobutan-2-ylcarbamate (137 was heated at 130°C. overnight. The mixture was cooled to rt, mg, 25%). diluted with water and extracted with EtOAc. The organic "H NMR (500 MHz, CDC1) 8 ppm 0.93 (s, 9H) 1.44 (s, 65 phase was washed with brine, dried over NaSO and evapo 9H) 5.12 (d. 1H) 5.40 (d. 1H) 7.46 (d. 2H) 7.95 (d. 2H). MS rated. The product was purified on a silica column (EtOAc: (ESI) m/z 325.8 M+H". hexane 1:10) to give tert-butyl 1-(4-cyanophenyl)-3,3-dim US 8,859,556 B2 13 14 ethyl-1-oxobutan-2-ylcarbamate (96.1 mg, 43%). tert-Butyl glass filter (P3 sintered disk 100 mm diameter), washed with 1-(4-cyanophenyl)-3.3-dimethyl-1-oxobutan-2-ylcarbamate ice-cold MeTHF (100 mL). The product was dried in vacuo at (96.1 mg, 3.04 mmol) was dissolved in 1.25 M HCl in MeOH 40° C. to give 101 g as a di-hydrochloride salt (65%). The at 0°C. and the mixture was stirred at rt for 8 h. The solvent purity was determined on GC giving 96.7 area-% and the was removed and the residue dried in vacuo to give 4-(2- NMR assay was 71.5% w/w (base). Yield 75% based on NMR amino-3,3-dimethyl-butanoyl)-benzonitrile hydrochloride assay. (110 mg, 93%). Final Compounds H NMR (400 MHz, MeOD) 8 ppm 1.02 (s, 9H) 5.05 (s, Example 5 1H) 7.96 (d. 2H) 8.20 (d. 2H). MS (ESI) m/z 217.1 M+H" Method 2 10 N-(1-(4-Cyanophenyl)-3,3-dimethyl-1-oxobutan-2- To a cleaned and dried 10L reactoratT,65°C. was charged yl)-4-(6-methylpyridin-2-yl)piperazine-1-carboxam 2-propanol (573 mL) and HCl in 2-propanol (5M; 696 mL: 5 ide eq). To the mixture at T, 53° C. was added portion-wise (approx 150 mL) the solution tert-butyl 1-(4-cyanophenyl)- 3.3-dimethyl-1-oxobutan-2-ylcarbamate from the previous 15 stage (Example 2b as described above) during 9 min (after 2 approx 600 mL charging the product starts to precipitated O out). The slurry was stirred for 1 hand analyses gave complete conversion (GC). The fine crystalline slurry was cooled (T,0° r N l N C.) and left overnight. N O The product was isolated on glass filters (2 filters: P3 N - H sintered disk; 130 mm diameter) in vacuo (slow filtration during approx3h). The product was displacement-washed in each filter with a mixture of cold (0°C.) 2-propanol (496 mL.; l 2.6 vol) and iPrOAc (650 mL: 3.4 vol) and with MeTHF 25 (2x425 mL: 2x3 vol). The product was dried in vacuo 40°C. over a weekend giving 135 g of white fine crystalline hydro Method 1 chloric acid salt. Analyses gave a HPLC purity of 99.5 area-% 4-(2-Amino-3,3-dimethylbutanoyl)benzonitrile hydro (254 nm) and a NMR assay of 94% w/w. Yield 72% based on chloride (Example 3, 50 mg, 0.23 mmol) was added to a NMR assay over two stages. 30 solution of CDI (112 mg 0.69 mmol) and TEA (0.035 mL, 0.25 mmol) in MeCN (5 mL). The reaction mixture was Example 4 stirred at rt for 90 minutes before 1-(6-methylpyridin-2-yl) piperazine (CAS 55745-89-6, 205 mg, 1.16 mmol) was 1-(6-Methylpyridin-2-yl)piperazine added. The mixture was stirred for 1 h and the solvent was evaporated under reduced pressure. The product was dis 35 solved in methanol, filtered and purified by prep. HPLC yield ing N-(1-(4-cyanophenyl)-3.3-dimethyl-1-oxobutan-2-yl)-4- (6-methylpyridin-2-yl)piperazine-1-carboxamide (36.3 mg, 37%). H NMR (400 MHz, CDC1) 8 ppm 0.94 (s, 9H) 2-40 (s, 40 3H)3.48-3-68 (m, 8H) 5.25-5-31 (m, 1H) 5.33-5.39 (m, 1H) 6.43 (d. 1H) 6.53 (d. 1H) 7.40 (dd. 1H) 7.78 (m, 2H)8.14 (m, 2H). MS (ESI) m/z 418.3 M-H Method 2 To a cleaned and dried /2 L round-bottom flask was added Bis(trichloromethyl)carbonate (60.6 mg, 0.20 mmol) was 2-chloro-6-methylpyridine (80 g; 620 mmol; 1.0 eq) and pip 45 added portion-wise to 4-(2-amino-3,3-dimethylbutanoyl) erazine (400 g, 7.40 eq). The mixture was heated using an benzonitrile hydrochloride (Example 3, 129 mg, 0.51 mmol) external oil bath (T, 154° C) and magnetically stirred. After and TEA (0.285 mL 2.04 mmol) in DCM (2 mL) under 1 approx 6 h reaction time the reaction was considered to be minute at rt. After stirring for 20 min, a solution of 1-(6- complete (GC analyses) and allowed to cool to room tem methylpyridin-2-yl)piperazine (CAS 55745-89-6, 90 mg. perature. To the cold mixture was added toluene (475 mL: 6 50 0.51 mmol) and TEA (0.142 mL, 1.02 mmol) in DCM (2 mL) Vol) and water (633 mL. 8 Vol) giving two clear phases. The was added drop-wise and the reaction was stirred for 90 min. water phase was recovered and extracted with toluene (150 The volatiles were removed before the reaction mixture was mL). The toluene layers were combined, washed with brine diluted with MeOH, filtered and purified by preparative (17.5% w/w: 150 mL) and concentrated in vacuo to give an HPLC. Fractions were pooled and freeze-dried to give N-(1- oily residue (estimated amount 88 g free base). To a 1 L (4-cyanophenyl)-3,3-dimethyl-1-oxobutan-2-yl)-4-(6-meth reactor at room temperature was added a solution of free base 55 ylpyridin-2-yl)piperazine-1-carboxamide (76 mg, 36%). dissolved in MeTHF (790 mL. 10 vol) to an aq. HCl solution H NMR (500 MHz, MeOD) 8 ppm 0.99 (s.9H) 2.36 (s, (1N: 396 mL; 5 eq). The yellow water phase was recovered 3H)3.53 (m, 8H) 5.30 (m. 1H)6.34 (m, 1H) 6.57 (t,3H) 7.44 and cooled with an external ice-water bath and basified to (dd. 1H) 7.88 (m, 2H) 8.17 (m, 2H). MS (ESI) m/z 420.2 pH>11 with NaOH (5M; 95 mL). The basic aq. phase was M+H" extracted with MeTHF (792 mL. 10 vol), the water phase was 60 Method 3 recovered and the organic layer was concentrated in vacuo To a cleaned and dried 10 L reactor at T, 22°C. was added and dried by azeotropic distillation. The dried MeTHF prod CDI (153.7g: 0.74 mol; 1.5 eq) followed by DMF (1 L; 8.0 uct-containing layer (470 mL. 5 Vol) was cooled with an Vol) giving a clear pale yellow solution. To the solution above external ice-water bath giving an opaque Solution. To the was added at T, 22°C., 10 portions of a pre-made suspension/ solution was slowly added HCl in 2-propanol (310 mL, 3.0 65 solution of 4-(2-amino-3,3-dimethylbutanoyl)benzonitrilex eq) and an off-white product precipitated out. The obtained HCl (Example 3 prepared by method 2: 135 g.: 0.50 mol; 1.0 slurry was cooled for 1 h and the product was isolated on a eq), DMF (1 L; 8.0 vol) and TEA (127 g; 1.26 mol) during 30

US 8,859,556 B2 17 18 slit and a programmable anti-scatter slit giving an irradiated Biological Evaluation length of 10 mm were used. 0.02 radian Soller slits were used Assay for Determining Biological Activity on the incident and on the diffracted beam path. A 20 mm The assay was designed to detect compounds that act on fixed mask was used on the incident beam path and a Nickel hTRPA1 by monitoring intracellular Ca" levels in whole filter was placed in front of a PIXcel-detector using 255 active cells. To this end, a dual addition step FLIPR (Fluorescence channels. Thin flat samples were prepared on flat silicon Zero Imaging Plate Reader) assay for TRPA1 activity has been background plates using a spatula. The plates were mounted designed. Briefly, HEK293 cells expressing TRPA1 are in sample holders and rotated in a horizontal position during grown in a 384 well microtitre plate and loaded with Fluo-4, measurement. Diffraction patterns were collected between 2 a fluorescent probe that reports changes in intracellular cal 10 cium. TRPA1 channel activity is assayed by measuring a 2theta and 40° 2theta in a continuous scan mode. Total time baseline signal in assay buffer, followed by application of an for a scan between 2 and 40° 2theta was approximately 10 ECs-concentration of the TRPA1-agonist zinc. The subse minutes. quent influx of calcium through TRPA1 channels is detected A diffractogram for (R) N-(1-(4-cyanophenyl)-3,3-dim as a rise in cytoplasmic calcium, which in turn is reported as ethyl-1-oxobutan-2-yl)-4-(6-methylpyridin-2-yl)piperazine 15 an increase in the Fluo-4 fluorescence. The activity of test 1-carboxamide is shown in FIG. 1. compounds is assessed by adding compounds five minutes Vibrational Circular Dichroism prior to the addition of zinc. TRPA1 blockers (antagonists) Computational Spectral Simulations: inhibit the calcium influx elicited by the Zinc addition and A Monte Carlo molecular mechanics search for low energy hence no increase in Fluo-4 fluorescence occurs. TRPA1 geometries was conducted for the Senantiomer fragment (II) openers (agonists) by themselves give rise to calcium influx using MacroModel within the Maestro graphical interface and an increase in the Fluo-4 fluorescence detected immedi (Schrödinger Inc.). All conformers within 5 kcal/mole of the ately after compound addition. lowest energy conformer were used as starting points for In the assay expression of TRPA1 in the HEK293 cell line density functional theory (DFT) minimizations within Gaus is under the control of an inducible promoter. Therefore, it is sian03. possible to establish the specificity of the TRPA1 signal by 25 comparing the signal from the Zinc stimulus on induced and non-induced cells. 2 TRPA1 is activated by a number of irritants that cause pain. O Zinc is an essential biological trace element that excites noci ceptive sensory neurons in mice in a TRPA1-dependent mat 30 ter. Zinc activates TRPA1 through a unique mechanism that r H requires Zinc influx through TRPA1 channels and subsequent activation via specific intracellular cysteine and histidine resi itsu O dues. hTRPA1-HEK293-TREx cells were seeded in Poly-D- (II) Fragment for which VCD calculations were per Lysine coated plate and allowed to grow a confluent mono formed 35 layer in cell medium. Prior to the experiment the medium was Optimized structures, harmonic vibrational frequencies/ discarded and cells loaded with fluo-4 NW (Molecular intensities, VCD rotational strengths, and free energies at Probes) in assay buffer for 1 hour in room temperature. Com STP (including Zero-point energies) were determined for pounds were added to the cell-plate and pre-incubated for 5 each conformer. In these calculations, the B3PW91 general minutes in assay buffer without Ca". 200 mM zinc in assay ized gradient approximation (GGA) exchange-correlation 40 buffer with Ca" was subsequently added to the cells and raw density functional was used in conjunction with the 6-31G fluorescent counts measured using excitation LED-banks basis set. Simulation of VCD spectra for each conformation with wavelength 470-495 nm and emission filter with wave were generated and fit to Lorentzian line shapes (12 cm line length 515-575 nm. The typical assay conditions were: Test width), and the computed spectra were Boltzmann-weighted compounds: 30 uM to 0.001 uM, or zero in positive and to allow direct comparisons between simulated and experi 45 negative controls; Assay buffer pH 7.4: HBSS with or without mental spectra. Ca" and Mg", 10 mM Hepes, 1 mM Glucose, 0.4% VCD Experimental: NaHCO, Agonist; 200 uM Zinc chloride, Compounds to be The experimental spectra were obtained by dissolving -8 tested were diluted in 100% DMSO and prior to the experi mg of sample in -0.15 mL of CDC1. Analyses were con ment further diluted 50 times in assay buffer. ducted at 4 cm resolution in a 0.1 mm BaFi cell using the 50 In the assay 100% activity was defined as the peak fluores dual source, dual PEM, VCD scan protocol using the cence level caused by 200 uM zinc in absence of test com BioTools ChiralIR instrument. The instrument incorporated a pound. ICsos represented the concentration of test compound dual photo-elastic modulator set for polarization modulation required to inhibit this response with 50%. at 37.024 kHz with W4 retardation (optimized for acquisition Data from this assay for exemplified compounds is shown of the spectral region centered around 1300 cm). Lock-in in the table below. The potency is expressed as ICso (concen amplification with a 30 us time constant, and a 20 kHz, high 55 tration needed for 50% inhibition relative to the mean signal pass and a 4 kHz, low pass filter was used. in calcium buffer) and the value indicated is an average of at VCD Results: least two individual experiments. FIG. 2 shows 4 spectra. Spectrum (1) is the experimentally collected spectrum of example 6. Spectrum (2) is a calculated TABLE 1 60 estimation of the (R) enantiomer VCD spectrum which was ICso values for the final compounds obtained via inversion of the ab-initio calculated spectrum of fragment (II). Spectrum (3) corresponds to the experimental Example Mean ICso (IM) VCD spectrum of example 7. Spectrum (4) is the calculated 5 (racemate) O.2 spectrum for fragment (II), which is the (S)-enantiomer. 6 (R-enantiomer) O.O6 Spectra 1 and 2 in FIG. 2 provide assignment of example 6 as 65 7 (S-enantiomer) 6.8 the (R) enantiomer. The analogous comparison of Spectra 3 and 4 indicates example 7 to exist as the (S) enantiomer. US 8,859,556 B2 19 20 What is claimed is: 3. A pharmaceutical composition comprising a compound 1. A compound N-(1-(4-cyanophenyl)-3,3-dimethyl-1-ox obutan-2-yl)-4-(6-methylpyridin-2-yl)piperazine-1-car according to claim 2 and a pharmaceutically acceptable car boxamide of formula (I) rier. 5 (I) 4. A method for the treatment of asthma, pertussis, nicotine addiction, acute pain disorders, or chronic pain disorders in a 2. Subject in need of such therapy, comprising administering to O said subject atherapeutically effective amount of a compound 10 according to claim 2. r N l NH 5. A method for the treatment of pain in a subject in need of N N - O Such therapy, comprising the step of administering to said Subject a therapeutically effective amount of a compound 2N 15 according to claim 2. 6. The method of claim 4, wherein the acute pain disorder or a pharmaceutically acceptable salt thereof, enantiomer, or chronic pain disorder is widespread pain, localized pain, or mixture thereof. 2O 2. A compound according to claim 1 that is (R)-N-(1-(4- nociceptive pain, inflammatory pain, central pain, central and cyanophenyl)-3,3-dimethyl-1-oxobutan-2-yl)-4-(6-meth peripheral neuropathic pain, central and peripheral neuro ylpyridin-2-yl)piperazine-1-carboxamide having the follow genic pain, central and peripheral neuralgia, chronic tendini ing formula: tis, low back pain, postoperative pain, peripheral neuropathy, 2 25 visceral pain, pelvic pain, allodynia, anesthesia dolorosa, O causalgia, dysesthesia, fibromyalgia, hyperalgesia, hyperes thesia, hyperpathia, ischemic pain, Sciatic pain, pain associ 1s, ated with cystitis, pain associated with multiple Sclerosis, N O pain associated with arthritis, pain associated with osteoar N thritis, pain associated with rheumatoid arthritis, or pain asso ciated with cancer. l 7. The method of claim 6, wherein the cystitis is interstitial 35 cystitis. or a pharmaceutically acceptable salt thereof.